Proteins are macromolecules that perform maintenance and function of life phenomena, and are responsible for a wide range of biological roles among various materials present in vivo. In order to perform these roles, protein-protein interactions should first be achieved, which is considered to be the basis of all life phenomena. If the protein interactions are not properly regulated, homeostasis in the body is destroyed, resulting in various diseases. Accordingly, various methods for treating diseases by artificially regulating the protein interactions have been studied and developed, and various pharmaceutical products that are actually usable for therapeutic purposes have been successfully developed.
Antibodies are important proteins of an immune response, and perform biological functions through specific interaction with antigens. This specific binding capacity enables a high therapeutic effect without side effects unlike conventional low molecular chemical agents. Therefore, many research institutions and pharmaceutical companies are actively conducting research to develop the antibodies having binding capacity with respect to well-known therapeutic agent targets using a variety of screening techniques. Due to the low side effects and high therapeutic efficacy compared to the chemical agents, the development of antibody therapeutic agents have been heavily invested by global pharmaceutical companies and biotechnology companies, and thus, a number of antibody therapeutic agents are currently used in clinical practice, and many therapeutic candidates are in clinical trials. In addition, it is widely used in various fields such as separation and purification of biomaterials and molecular medical diagnosis technology, etc., as well as for therapeutic purposes. However, in spite of these advantages, there are problems in that production cost is high, it is difficult to escape the existing patent barriers, it is difficult to penetrate into cells due to large molecular weight, and therapeutic effects of patients are not actually high as expected. Accordingly, in recent years, development of artificial antibodies for replacing the antibody therapeutic agents has been actively conducted. A number of researches have revealed that these artificial antibody scaffold proteins are largely improved in efficiency of penetration into cancer tissues unlike the antibody therapeutic agents, and as a result, therapeutic effects are able to be improved.
Under these circumstances, the present inventors have successfully developed a repebody which is a non-antibody protein scaffold capable of replacing the conventional antibodies. The repebody refers to a polypeptide optimized by consensus design through fusion based on similarity between an N-terminal of internalin and a variable lymphocyte receptor (VLR) structure having a leucine-rich repeat (LRR) structure. The repebody has a size that is about ⅕ a size of the antibody, and is mass-produced in Escherichia coli, and has almost no immunogenicity as a result of animal tests. Further, the repebody has significantly excellent stability against heat and pH, and is able to very easily increase binding capacity to a target up to a pico-mole level, and has remarkably superior specificity to the target.
Meanwhile, the complement protein C5a is known to be a disease inducer causing immune diseases such as asthma, rheumatoid arthritis, and lupus. In particular, the complement protein C5a receives attention as a main target in the treatment of sepsis, which is a disease that shows addition or causes an infection in the whole body due to toxic materials produced by propagation and reproduction of various germs in blood. The sepsis is known worldwide as a major cause of intensive care unit (ICU) morbidity and mortality. In the United States, there are 750,000 sepsis patients each year, and in Korea, very high incidence is shown even though there is no accurate statistics. The mortality rate is also very high, about 40%. However, a therapeutic agent which was FDA-approved in 2001, was withdrawn from the market in 2011, and there are still very few effective treatment methods or therapeutic materials. Therefore, research on the development of a therapeutic agent targeting the complement protein C5a as a therapeutic agent for sepsis has been actively conducted.
The present inventors have successfully produced a specific protein binder for various disease-related target proteins using the above-described repebody scaffold, and have verified to have a biological inhibition effect based on cell-based methods. However, this application study thereof is only in the beginning stage, and thus, further research is actively underway.
Therefore, the present inventors have made efforts to develop a protein that specifically binds to the complement protein C5a, which is known to be related to various immune diseases using the above-described repebody scaffold, and as a result, selected a novel polypeptide having a specific binding capacity to the complement protein C5a based on random mutation library constructed through modularity which is a structural characteristic of the repebody and overall structure analysis, and confirmed that the binding strength of the complement protein C5a to the polypeptide is higher than that of a complement protein C5a receptor which is present in nature, and completed the present invention.